Assembly and comparative analysis of the complete mitochondrial genome of Isopyrum anemonoides (Ranunculaceae)

Ranunculaceae is a large family of angiosperms comprising 2500 known species—a few with medicinal and ornamental values. Despite this, only two mitochondrial genomes (mitogenomes) of the family have been released in GenBank. Isopyrum anemonoides is a medicinal plant belonging to the family Ranunculaceae, and its chloroplast genome has recently been reported; however, its mitogenome remains unexplored. In this study, we assembled and analyzed the complete mitochondrial genome of I. anemonoides and performed a comparative analysis against different Ranunculaceae species, reconstructing the phylogenetic framework of Isopyrum. The circular mitogenome of I. anemonoides has a length of 206,722 bp, with a nucleotide composition of A (26.4%), T (26.4%), C (23.6%), and G (23.6%), and contains 62 genes, comprising 37 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and three ribosomal RNA (rRNA) genes. Abundantly interspersed repetitive and simple sequence repeat (SSR) loci were detected in the I. anemonoides mitogenome, with tetranucleotide repeats accounting for the highest proportion of SSRs. By detecting gene migration, we observed gene exchange between the chloroplast and mitogenome in I. anemonoides, including six intact tRNA genes, six PCG fragments, and fragments from two rRNA genes. Comparative mitogenome analysis of three Ranunculaceae species indicated that the PCG contents were conserved and the GC contents were similar. Selective pressure analysis revealed that only two genes (nad1 and rpl5) were under positive selection during their evolution in Ranunculales, and two specific RNA editing sites (atp6 and mttB) were detected in the I. anemonoides mitogenome. Moreover, a phylogenetic analysis based on the mitogenomes of I. anemonoides and the other 15 taxa accurately reflected the evolutionary and taxonomic status of I. anemonoides. Overall, this study provides new insights into the genetics, systematics, and evolution of mitochondrial evolution in Ranunculaceae, particularly I. anemonoides.


Introduction
Isopyrum belongs to the family Ranunculaceae.The genus contains four species, mainly distributed in the temperate regions of the Northern Hemisphere [1].Two species, I. manshuricum and I. anemonoides, are found in China [2,3].Isopyrum plants contain various chemical components, including benzylisoquinoline alkaloids, flavonoids, triterpenoid saponins, cyanides, and lactones, and most of its genera are used in traditional Chinese medicine to clear away heat and detoxification, relieve swelling and pain, and treat cardiovascular diseases [4].For example, the root uber of I. anemonoides is highly valued for its medicinal properties [5].While the chloroplast genome of I. anemonoides has been recently reported, its mitochondrial genome (mitogenome) remains unexplored.
The mitochondrion is a semi-autonomous organelle with its own genetic material and genetic system, providing a source of chemical energy for eukaryotes through oxidative phosphorylation [6].The typical angiosperm mitogenome is a double stranded DNA molecule, and its size is extremely variable between different species, ranging from 66 kb (Viscum scurruloideum) to over 11.3 mb (Silene conica), even among closely related species [7,8].Angiosperm mitochondrial genomes are known for their low mutation rate, relatively high incidence of RNA editing and trans-splicing of coding sequences, and dynamic structure [9].Most size and structural variations in angiosperm mitogenomes are related to the uptake of foreign sequences because the mitochondrial genes are obtained from gene transfer between nuclear and plastid genomes or even different species [10,11].Consequently, the assembly of mitogenomes can be more complex and challenging than that of chloroplast genomes [12].With the development of next generation sequencing technology, obtaining mitogenome sequences have become more feasible [13][14][15][16].Several studies have indicated that mitogenomes are popular genetic markers for population genetics, molecular ecology, plant classification, and evolution [17,18], such as in Ginkgo and Welwitschia [19], Oleaceae [20], Dioscoreales [21], and Vitaceae [22].Despite comprising 2500 known species, data on the mitochondrial genomes of the Ranunculaceae family are lacking, with only two mitochondrial genomes released in Gen-Bank as of December 2022.This limitation hinders the overall understanding of the phylogenetics and adaptive evolution of Ranunculaceae species [23,24].
Given the importance of the plant mitochondrial genome in understanding the phylogenetics and adaptive evolution of Ranunculaceae species, this study aims to sequence and analyze the complete mitogenome of I. anemonoides and compare it with the mitogenomes of two other Ranunculaceae species, Aconitum kusnezoffii (NC053920) [23], and Anemone maxima (MT568500) [24].This study will contribute to exploring the structure and evolution of mitogenomes, and highlight the need for more mitogenomes to be sequenced in the family Ranunculaceae.

Plant materials, DNA sequencing and assembly
Fresh leaves of I. anemonoides were collected from Jimusaer County in Xinjiang Autonomous Region, China (89˚18' N, 44˚00' E).The plant material was identified by Xinmin Tian (http:// sky.xju.edu.cn/info/1151/1956.htm)using the detailed species information in Flora of China (http://www.iplant.cn/foc).The samples were immediately frozen with silica gel and stored at −20˚C.As a non-protected plant, collection of the I. anemonoides is in accordance with the Laws of China's National Forests and Grasslands Administration (no sampling license is required).Voucher specimens (TXM202006) were deposited at the Herbarium of Xinjiang University (XJU).All methods used in this study were carried out in accordance with relevant guidelines and regulations.
The total genomic DNA was extracted using the Plant Genomic DNA Kit (China Tiangen Biotechnology, Beijing, China) and then sequenced using the Illumina Hiseq 2000 platform (Illumina, San Diego, CA, USA).Raw data from paired-end sequencing was filtered to eliminate low-quality sequences, sequences with high "N" ratios, fragments with lengths less than 25 bp, and unknown nucleotides.The clean data produced a total of 6.13 Gb/6.10 Gb.The high-quality clean reads were assembled using GetOrganelle v.1.7.5 with default settings and k-mer values set to 21, 43, 65, 87, and 127 [25].Contigs were selected using BLAST software (query coverage � 70% and E-value � 1e-10) with the mitogenome of Aconitum kusnezoffii (NC053920) as the reference.Geneious Prime 2022.1.1 (https://www.geneious.com)(Biomatters Ltd., Auckland, New Zealand) was used for mitochondrial contig mapping and alignment to verify quality with custom sensitivity (1% maximum for each gap and mismatches allowed).The final mitogenome was obtained using the Geneious mapper and aligner.

Identification of mitochondrial plastid DNAs (MTPTs)
To identify plastid-derived DNA fragments in the mitogenome, we compare the plastome of I. anemonoides with the mitogenome.The plastid genome data of I. anemonoides was obtained from our previous study (OM457045) [32].We used Tbtools v.0.668 (https://www.tbtools.com/) [33] to identify sequences shared between the plastid and mitogenome with screening criteria set to a matching rate of � 70%, E-value of � 1e-5, and length of � 40.

Selective pressure analysis and prediction of RNA editing sites
We calculated the non-synonymous substitution rate (dN) and synonymous substitution rate (dS) of each PCG in I. anemonoides, A. kusnezoffii, and A. maxima of the Ranunculaceae family.First, the PCGs of these species were extracted using PhyloSuite v.1.2.2 [34] (http:// phylosuite.jushengwu.com/) and separately aligned using MAFFT v.7.407 [35], with all stop codons removed.The alignments were manually checked for accuracy.We then used the yn00 module in PAML v.4.9 [36] to calculate the dN and dS values, estimating pairwise nucleotide substitution rates with the following parameters: verbose = 0; icode = 0; weighting = 0; com-monf3 × 4 = 0 (use one set of codon freqs.for all pairs); ndata = 1dN/dS.For RNA editing site analysis, all mitogenome PCGs of the three species were extracted using the same approaches as above, and RNA editing sites were predicted using PREP-Mt (http://prep.unl.edu/)[37], with a cutoff value of 0.2.

Features of the I. anemonoides mitogenome
The complete circular mitogenome of I. anemonoides was 206,722 bp in length, with the typical structure observed in most land plants ( Fig 1).The nucleotide composition of the mitogenome was determined to be 26.4% A, 26.4% T, 23.6% G, and 23.6% C, resulting in a GC content of 47.2% and an AT content of 52.8%.A total of 61 genes were identified in the mitogenome of I. anemonoides, comprising 37 PCGs, three ribosomal RNA (rRNA) genes, and 21 transfer RNA (tRNA) genes, with 20 exons and 12 introns (Table 1).The PCGs were classified into nine categories: NADH dehydrogenases (nine genes), succinate dehydrogenases (one gene), ubiquinol cytochrome c reductases (one gene), cytochrome c oxidases (three genes), ATP synthases (five genes), ribosomal proteins (10 genes), maturases (one gene), transport membrane proteins (one gene), and cytochrome c biogenesis (four genes) (S2 Table ).
The 37 PCGs varied from 222 bp (rps10) to 7,322 bp (nad4), with a total length of 51,185 bp, accounting for 24.76% of the I. anemonoides mitogenome.The lengths of tRNA and rRNA were 1,497 bp and 1,149 bp, making up 0.72% and 0.56% of the mitogenome, respectively (Table 1).Among the seven intron-containing genes in the I. anemonoides mitogenome, nad7 has three introns, whereas rps3, nad5, and ccmFC have one intron each.In addition, nad2, nad4, and nad1 have two introns each.The introns in the nad1 gene have lengths of 40,561 bp and 35,117 bp (S2 Table ).

Codon usage analysis of PCGs
Most PCGs in the I. anemonoides mitogenome had the typical ATG start codon, except for atp6 and mttB, which had TTG and CTG as their start codons, respectively (S2 Table ).Leucine (Leu) was the most frequently used amino acid.At the same time, methionine (Met) and tryptophan (Trp) were the least used amino acids among the 37 PCGs.We calculated the RSCU of the 37 PCGs in the I. anemonoides mitogenome and observed that the RSCU values of 31 codons were greater than 1.00 (Fig 2).These results indicate a strong bias toward a high representation of NNA and NNT, similar to other land plant species.

Repeat sequence analysis
Repeat sequence analysis of the I. anemonoides mitogenome revealed a total of 507 interspersed repetitive sequences (>30 bp), including 261 palindromic (67,304 bp) and 246 forward (47,398 bp); no complementary or reverse repeats were observed (Fig 3).The total length of the dispersed repetitive sequences was 114,702 bp, which accounted for 55.49% of the I. anemonoides mitogenome.These repeats ranged from 31 to 1,624 bp (nine were longer than 1 kb) (S3 Table ).Most of these dispersed repeats were observed in the nad1 introns and gene spacer regions, and a few were located in the gene coding region.Additionally, 52 tandem repeats   ).Most of these tandem repeats were observed in two copies, with 21% having a length of 0-50 bp, 29% with a length of 51-100 bp, and 50% with a length larger than 100 bp (S4

Identification of MTPTs
In this study, we observed 15 DNA fragments shared between the chloroplast and mitogenomes with a total length of 7,302 bp, accounting for 3.53% of the mitogenome (Table 2).These fragment lengths ranged from 59 to 1,576 bp, and the sequences exhibited more than 95% similarity in the mitochondrial and chloroplast genomes.Among these shared fragments, six were intact tRNA genes, six were partial PCGs, and the remaining fragments were part of the chloroplast ribosome RNA, namely trnW-CCA, trnP-UGG, trnD-GUC, trnN-GUU, trnM-CAU, trnT-GGU and psbB, petL, petG, rps14, psbD, ndhF, rrn16, and rrn23 (Table 2).

Comparison of mitogenome sizes and GC contents between I. anemonoides and the other two species from Ranunculaceae
We compared the genome sizes, GC contents, PCGs, rRNAs, and tRNAs of I. anemonoides with those of other published Ranunculaceae mitogenomes, including Anemone maxima and Aconitum kusnezoffii (Table 1).Their GC content was similar, ranging from 46.2% to 47.2% (Fig 4).However, their mitogenome sizes varied greatly, with A. maxima having the largest mitogenome at 1122,546 bp, followed by A. kusnezoffii at 440,720 bp and I. anemonoides at 206,722 bp.These three plants have similar proportions of rRNA genes.However, the proportions of tRNA and PCGs are significantly different among families (Table 1).This variation may be attributed to differences in mitogenome sizes.

Gene duplications and losses in the mitogenomes of the three Ranunculaceae species
Mitogenome loss is a common phenomenon in land plants; therefore, the fate of mitogenome loss has received increasing attention.However, certain genes, such as the Complex I gene (nadX), Complex III gene (cob), Complex IV gene (cox1-3), Complex V gene, and ccmX gene, are relatively conserved.In contrast, other types of genes may be lost in different plants (Table 3).Here, we compared the PCGs of I. anemonoides, A. maxima, and A. kusnezoffii.These three species have 37, 39, and 37 PCGs, respectively.As shown in Table 3, PCG loss events occurred in the mitogenomes of these species.For example, the atp1 gene was duplicated three times in I. anemonoides.In addition, the sdh3 and rps1 genes were lost compared to the other two species.A. maxima has an almost complete mitogenome, with only two genes (rps10 and rps14) lost.In contrast, the rps2, rps11, rps19, and rpl2 genes were lost from the A. kusnezoffii and I. anemonoides mitogenomes.In addition, the number of rRNA genes in the three species was the same; however, the number and type of tRNA genes differed.

Prediction of RNA editing sites in PCGs
RNA editing is a process that occurs in the mitochondria and plastids of plants.In plant mitogenomes, RNA editing changes the genomic information by converting cytosine (C) to uridine (U).In this study, we predicted the RNA editing sites of 33 common PCGs in the mitogenomes of three Ranunculaceae species.Our results showed that the number of RNA editing sites predicted in the I. anemonoides, A. maxima, and A. kusnezoffii mitogenomes was 554, 611, and 605, respectively.Furthermore, all editing sites in the three species occurred only at the first and second positions of the triplet codes, with none observed at the third position.Among the 33 PCGs, the ccmFn, ccmB, ccmC, and nad4 genes had the most editing sites (Fig 5 ), whereas few RNA editing sites were observed in rpl10, rps7, and sdh4.

Substitution rates of protein-coding genes
The pairwise dN/dS ratio can be used to determine the presence of selective pressure on specific PCGs during evolution.In general, dN/dS = 1 represents neutral selection, while dN/dS >1 or dN/dS <1 represents positive or negative selection, respectively.In this study, the pairwise dN/dS ratio was determined for 32 PCGs common to the I. anemonoides, A. maxima, and A. kusnezoffii mitogenomes.The nad3 gene was conserved in all three species and was thus excluded from this analysis.The results showed that most dN/dS ratios were less than 1.0, mainly for atp1, atp9, nad2, nad4L, nad6, rpl16, rps12, and cob, suggesting that most of the PCGs were subject to negative selection during their evolution (Fig 6 and S6 Table ).In contrast, the dN/dS ratios of ccmB, cox2, nad1, rpl5, and rps1 were greater than 1.0 in the I. anemonoides and A. kusnezoffii mitogenomes.In addition, the dN/dS ratios of atp4, atp6, cox2, cox3, matR, nad1, rpl5, rps7, and sdh4 genes were greater than 1.0 in the I. anemonoides and A. maxima mitogenomes (Fig 6).Only two genes, nad1 and rpl5, had dN/dS ratios greater than 1.0 in all three species, which indicated these genes were under positive selection during their evolution.

Phylogenetic analysis
In this study, a phylogenetic analysis was conducted on I. anemonoides and 15 other species, including 11 dicotyledonous, three monocots, and one gymnosperm (designated as outgroups).Maximum likelihood (IQ-TREE) and Bayesian (MrBayes) analyses produced identical topologies, strongly supporting the separation of dicotyledonous from monocots and the separation of angiosperms from gymnosperms with a high support value.I. anemonoides, A. kusnezoffii, and A. maxima formed a closely-clustered branch with high confidence (100% SH-aLRT support, 100% ultrafast bootstrap support in ML, posterior probability 100% in BI).
trnQ-UUG( 4 These findings provide valuable insights into the evolutionary history of I. anemonoides and serve as a foundation for further research.

Discussion
The mitochondrion is a semi-autonomous organelle that contains its own genetic material and genetic system, and it provides most of the energy for eukaryotes [40].Mitogenomes in higher plants vary significantly in size and complexity within families and genera.For example, Cucumis melo (2.9 Mt) and Citrullus lanatus (379,236 nt) [41], and Silene conica (11,318,806 nt) and S. latifolia (253,413 nt) [42,43].Due to this size variation and their complex structure, which includes frequent intra-genomic rearrangement, repeat sequences, gene transfer/loss, and highly variable levels of RNA editing, plant mitogenomes are more complex than those of animals [44,45].In this study, we sequenced and annotated the mitogenome of I. anemonoides and conducted a comparative analysis with two species from the Ranunculaceae family, A. maxima and A. kusnezoffii.The GC content of the three species was similar, ranging from 46.2% to 47.2%.However, the size of the mitogenomes varied greatly (Table 1).PCGs account for 24.76% of the I. anemonoides mitogenome, with the atp1 gene having three copies.PCGs play important roles in the plant mitogenome.For example, the mitochondrial atp1 gene, which encodes the protein ATP1 (α-subunit of mitochondrial ATP synthase F 1 ), is expressed differentially between sterile male lines in wheat [46].The gene cox1 is involved in the fertility transformation of the thermosensitive male-sterile line YS3038 in wheat [47].Moreover, similar to other angiosperms, most PCGs in the I. anemonoides mitogenome use the typical ATG start codon [48,49], whereas atp6 and mttB genes use TTG and CTG as start codons, respectively, which are modified by RNA editing.Repeat sequences are fragments that occur at multiple locations in the genome and can contain much genetic information, which is useful for developing markers for population and evolutionary analyses [50][51][52].In this study, we confirmed the presence of interspersed, tandem, and SSR repeats in the I. anemonoides mitogenome.Among them, dispersed repetitive sequences were the most prevalent, accounting for 55.49% (114,702 bp) of the I. anemonoides mitogenome (Fig 3).However, the repeat types found in the I. anemonoides mitogenome are different from those found in higher plants, such as Mangifera persiciformis (750,898 bp), M. longipes (728,635 bp), and M. sylvatica (714,426 bp) [53].Differences in the size of plant mitogenomes can be explained by variations in the size and type of repeat sequences present in mitochondrial genomes [54].
In plant genomes, gene transfer from chloroplasts to mitogenomes is common [55].Previous research has shown that gene transfer from organelle genomes to the nuclear genome is the most common direction in angiosperms, followed by transfers from the nuclear and plastic genomes to the mitogenome.In this study, we observed 15 gene fragments that have been transferred from the chloroplast genome to the mitogenome (Table 2).These sequences have a total length of 7,302 bp, accounting for 3.53% of the mitogenome.Among these, six complete tRNA genes were transferred, suggesting that they are more conserved than the PCGs and play an important role in the mitogenome [56,57].The transfer of genes from the chloroplast to the mitogenome may contribute to the high degree of rearrangements observed among mitochondrial genomes and promote genetic diversity, thus impacting eukaryotic evolution [58,59].
Additionally, we investigated gene loss events in the mitogenomes of I. anemonoides, A. maxima, and A. kusnezoffii (Table 3).Compared to higher plants, the mitochondrial genomes of these species exhibit relatively conserved genes, such as Complex I, Complex III-Complex V, rRNAs, transport membrane proteins, and maturases.Among the three species, A. maxima, has a considerably complete mitogenome, with only two genes (rps10 and rps14) lost.In contrast, rps2, rps11, rps19, and rpl2 genes were lost from the A. kusnezoffii and I. anemonoides mitogenomes.Moreover, the numbers and types of tRNA genes differed.Mitogenomes contain a large number of non-coding sequences and repeat sequences, and during evolution, gene substitution and functional transfer occur, leading to a considerable loss and transfer of genes.Typically, the missing functional genes in plant mitogenomes are compensated by the nuclear genome [60].Furthermore, tRNAs in plant mitogenomes are frequently lost and replaced by chloroplast-origin tRNAs, which are eventually transported or transferred to the mitochondrial genome [41].Because of the rapid evolution of mitogenomes and the loss of functional genes, gene loss can vary significantly between species, even among those belonging to the same family or genus.
RNA editing is a process that modifies genetic information at the transcriptional RNA level, occurring in the chloroplast and mitogenomes of higher plants and contributing to protein folding [61].Many studies have shown that RNA editing of the mitogenome is closely related to certain cultivated plant traits, such as those in Sorghum bicolor [62,63].In this study, we estimated approximately 554 RNA editing sites in I. anemonoides, 611 RNA editing sites in A. maxima, and 605 RNA editing sites in A. kusnezoffii mitogenomes.Among the 33 PCGs, the ccmFn, ccmB, ccmC, and nad4 genes had the most editing sites (Fig 5).In contrast, few RNA editing sites were found in rpl10, rps7, and sdh4.Ribosomal protein genes have fewer RNA editing sites, while ccmB and ccmFN genes have more editing sites; this has also been observed in other plants, such as Brassica napus [64] and Oryza sativa [65].RNA editing can create start and/or stop codons, resulting in more conserved proteins with higher homology than other proteins, allowing for better expression of mitochondrial genes.For example, in this study, RNA editing modified the start codons of the atp6 and mttB genes to TTG and CTG, respectively.Other studies have also reported these events, such as in Acer truncatum [59] and Lycopersicon esculentum [66].
Furthermore, most mitogenomes are highly conserved and have undergone neutral and negative selections [55].The dN/dS analysis results showed that most of the I. anemonoides PCGs were under negative selection, indicating that the PCGs in the mitogenome are conserved across land plants [56].Similar negative selection patterns were observed in another herb, S. glauca [56].However, comparative analysis results indicated that the dN/dS values of two genes (nad1, and rpl5) were greater than 1.0 in all three species, suggesting that these genes were under positive selection during evolution (Fig 6).nad1 is one of the subunits of ATP synthase and is an important mitogenome in plant breeding.Studies have shown that cytoplasmic male sterility is closely related to the function of nad1 [67].Meanwhile, rpl5 is a ribosomal protein gene involved in gene transcriptional regulation [68].These two genes might have developed novel functions for stress resistance in Ranunculaceae plants under positive selective pressure.Finally, we analyzed the phylogenetic relationship between I. anemonoides and representative taxa based on mitochondrial genes.Phylogenetic tree analysis showed a clear taxonomic relationship among the taxa.I. anemonoides, A. kusnezoffii, and A. maxima formed one clade with high support values ( Fig 7).

Conclusion
In this study, we assembled and analyzed the complete mitogenome of I. anemonoides and performed extensive analyses based on the annotated nucleotide sequences, which is a significant milestone in Isopyreae mitogenome research.The circular genome comprises 61 genes: 37 PCGs, three rRNAs, and 22 tRNAs, and exhibits sequence repeats and MTPT fragment migration.Comparative analyses of genome size and GC content indicated that the gene order tended to be conserved among the three species, with 14 core PCGs.Additionally, dN/dS analysis based on code substitution showed negative selections in most PCGs across the three Ranunculaceae species, indicating mitogenome conservation during evolution.These results and the availability of the mitogenome of I. anemonoides provide resources for future studies of Isopyreae and related lineages, which is an important clade of Ranunculaceae for comparative genomics and evolutionary research.

Fig 2 .
Fig 2. Relative synonymous codon usage (RSCU) in the Isopyrum anemonoides mitogenome.Codon families are shown on the x-axis.The RSCU values represent the frequency of each codon usage relative to the expected frequency based on uniform synonymous codon usage.https://doi.org/10.1371/journal.pone.0286628.g002

Fig 3 .
Fig 3. Repeat analysis of the I. anemonoides mitogenome.The C1 circle shows the dispersed repeats connected with blue and pink arcs from the center going outward: The C2 circle shows the tandem repeats as short bars.The C3 circle shows the microsatellite sequences identified using MISA.https://doi.org/10.1371/journal.pone.0286628.g003